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Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s).
Published
by S. Karger AG, Basel
DOI:
10.1159/000464389
DOI: 10.1159/000464389
© 2017
The Author(s)
www.karger.com/cpb
online:March
March07,07,
2017
Published online:
2017
Published by S. Karger AG, Basel
and Biochemistry Published
www.karger.com/cpb
1255
Jin
et al.: Effects of LAQ824 on Porcine Cloned Embryos
Accepted: September 15, 2016
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution
for commercial purposes as well as any distribution of modified material requires written permission.
Original Paper
The HDAC Inhibitor LAQ824 Enhances
Epigenetic Reprogramming and In Vitro
Development of Porcine SCNT Embryos
Jun-Xue Jina Sanghoon Leea Anukul Taweechaipaisankula Geon A. Kima
Byeong Chun Leea,b
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National
University, 1 Gwanak-ro, Gwanak-gu, Seoul, bInstitute of Green Bio Science and Technology, Seoul
National University, Daehwa-myeon, Pyeongchang-gun, Gangwon-do, Korea
a
Key Words
Pig, Histone deacetylase inhibitor • Somatic cell nuclear transfer • Reprogramming • In vitro
development
Abstract
Background/Aims: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming
results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic
remodeling drugs have been used in attempts to improve in vitro development of porcine
SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone
acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine
SCNT embryos. Methods: LAQ824 treatment was supplemented during the culture of SCNT
embryos. The reprogramming levels were measured by immunofluorescence and quantified
by image J software. Relative expression levels of 18 genes were analyzed by quantitative
real-time PCR. Results: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24
h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment
enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global
DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear
and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression
of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a
and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3
and Bak) in blastocysts. Conclusion: Optimum exposure (100 nM for 24 h) to LAQ824 postactivation improved the in vitro development of porcine SCNT embryos by enhancing levels
of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.
© 2017 The Author(s)
Published by S. Karger AG, Basel
Porcine somatic cell nuclear transfer (SCNT) is a powerful technique for basic and
biomedical research [1]. Cloned pigs may provide suitable donor organs for regenerative
Byeong Chun Lee, DVM, Ph.D.
Department of Theriogenology and Biotechnology, College of Veterinary Medicine,
Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826 (Korea)
Tel.+ 82-2-880-1269, Fax +82-2-873-1269, E-Mail bclee@snu.ac.kr
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Introduction
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1256
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
medicine and xenotransplantation [2]. However, despite the fact that offspring of several
mammalian species have been produced via SCNT, the efficiency of porcine SCNT remains
very low, less than 5% [3]. This is generally attributed to abnormal reprogramming, such as
histone hypoacetylation and DNA hypermethylation, rather than to any genetic mutations
[4]. It is well known that the majority of the reprogramming of donor cell nuclei occurs in
the recipient oocyte cytoplasm or in the early embryo before embryonic genome activation
(EGA) [5, 6]. Histone acetylation, a type of epigenetic modification, which seems to be a key to
successful reprogramming, is controlled by the activity of two groups of enzymes, including
histone acetyltransferases (HATs) and histone deacetylase (HDACs) [7]. It occurs within
a short period of time, causing unwinding of the chromatin structure to increase nuclear
volume, facilitating the regulation of gene expression in SCNT embryos [8].
It has been demonstrated that a histone deacetylase inhibitor (HDACi) improves
the levels of acetylation in nuclear donor cells or early SCNT embryos [9]. Recently, there
were several reports that HDACi treatment positively regulates histone acetylation levels,
epigenetic reprogramming, gene expression, full term embryo development and DNA
methylation levels in a manner similar to these events taking place in in vitro fertilized
embryos. The development of porcine SCNT embryos was improved by treatment with HDACi,
such as trichostatin A (TSA) [10, 11], valproic acid (VPA) [12], scriptaid [13, 14], oxamflatin
[15], sodium butyrate (NaBu) [16], LBH589 [17], CUDC-101 [18], m-carboxycinnamic acid
bishydroxamide (CBHA) [3], PXD101 [19], suberoylanilide hydroxamic acid (SAHA) [20] and
PCI-24781 [21].
LAQ824 (Dacinostat, (E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl) ethyl]
amino]-methyl]phenyl]prop-2-enamide) is a structurally novel HDACi belonging to a class
of hydroxamic acid analogs known to inhibit class I, IIa, and IIb histone deacetylases [22,
23]. However, the effect of LAQ824 on in vitro development of SCNT embryos has not been
studied. Hence, in the present study porcine SCNT embryos were treated with LAQ824 to
investigate whether it improved their developmental competence.
The objective of this study was to find the optimum conditions for LAQ824 treatment
for improving in vitro development of porcine SCNT embryos, and to determine the global
acetylation levels of histone 3 at lysine 9 (H3K9), histone 4 at lysine 12 (H4K12), and
global DNA methylation levels of anti-5-methylcytosine (5-mC) using immunofluorescence,
at different stages of porcine SCNT embryos. Then, we analyzed the effects of LAQ824
treatment on gene expression related to histone acetylation, DNA methylation, development
and apoptosis in blastocyst.
Materials and Methods
Oocyte collection and in vitro maturation
Porcine ovaries were collected from pre-pubertal gilts at a local slaughterhouse and transported to
the laboratory at 30-32 °C. The contents of follicles 3–6 mm in diameter were recovered by aspiration with
an 18-gauge needle. Cumulus-oocyte complexes (COCs) were pooled and washed three times with tissue
culture medium (TCM)-199 (Invitrogen, Carlsbad, CA, USA), 2 mM of sodium bicarbonate, 10 mM N-[2Hydroxyethyl] piperazine-N'- [2-ethanesulfonic acid] (HEPES), 5 mM sodium hydroxide, 1% Pen-Strep
(Invitrogen), and 0.3% polyvinyl alcohol (PVA). Then, 50 COCs were placed into in vitro maturation (IVM)
medium comprising TCM-199 supplemented with 2 mM sodium pyruvate, 5 µl/mL insulin transferrin
selenium solution (ITS) 100X (Invitrogen), 0.57 mM cysteine, 10 ng/mL epidermal growth factor (EGF),
10% porcine follicular fluid (vol/vol), 10 IU/mL human chorionic gonadotropin (hCG), and 10 IU/mL
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The protocol for animal use was approved by the Institutional Animal Care and Use Committee of
Seoul National University (SNU-151019-4) in accordance with the Guide for the Care and Use of Laboratory
Animals of Seoul National University. LAQ824 was purchased from Selleck Chemicals (Houston, TX, USA).
Other chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO, USA),
unless otherwise stated.
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1257
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
equine chorionic gonadotropin (eCG). The selected COCs were incubated at 38.5 °C under 5% CO2 in 95%
humidified air for IVM. Following 22 h of maturation with hormones, the COCs were washed twice in fresh
IVM medium and then cultured in hormone-free IVM medium for an additional 22 h.
Donor cell preparation for SCNT
Porcine fibroblasts were isolated from ear tissue of a Korean native adult pig. The tissue was cut into
small pieces and cultured at 38 °C in an atmosphere of 5% CO2 in air in Dulbecco’s modified Eagle's medium
(DMEM; Gibco, culture medium) containing 10% fetal bovine serum (FBS; Gibco, culture medium) (v/v),
1 mM sodium pyruvate, and 100 IU/mL each of penicillin and streptomycin. Cells from passages 3 to 7
were used as donors for SCNT. A single cell suspension was prepared by standard trypsinization procedures
immediately before SCNT.
Somatic cell nuclear transfer
After IVM, COCs were denuded by gently pipetting with 0.1% hyaluronidase, and washed three times in
Tyrode's albumin lactate pyruvate (TALP) medium using mouth pipette. Denuded oocytes were incubated in
TALP containing 5 µg/mL Hoechst 33342 for 10 min and observed under an inverted microscope equipped
with epifluorescence. An oocyte was held with a holding micropipette and the zona pellucida was partially
dissected with a fine glass needle to make a slit near the first polar body. Enucleation was performed by
aspirating the first polar body and adjacent cytoplasm containing the metaphase II chromosomes with an
aspiration pipette in TALP medium containing 5 µg/mL cytochalasin B. A single donor cell was inserted into
the perivitelline space of an enucleated oocyte, and the couplets were equilibrated in fusion solution (0.28 M
mannitol solution containing 0.5 mM HEPES and 0.1 mM MgSO4), and then fused in a 20 µL droplet of fusion
solution with a single DC pulse of 1.2 kV/cm for 30 µs using an electrical pulsing machine (LF101; Nepa
Gene, Chiba, Japan). After 1 h, fused couplets were equilibrated with activation solution (0.28 M mannitol
solution containing 0.5 mM HEPES, 0.1 mM CaCl2 and 0.1 mM MgSO4), and then transferred to a chamber
containing two electrodes overlaid with activation solution, and activated with a single DC pulse of 1.5
kV/cm for 30 µs using a BTX ElectroCell Manipulator 2001 (BTX Inc., San Diego, CA). SCNT embryos were
washed three times with fresh porcine zygote medium-5 (PZM-5; Funakoshi Corporation, Tokyo, Japan),
and transferred into 30 µL in vitro culture (IVC) droplets covered with mineral oil, and then cultured at 38.5
°C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2. To count total cell numbers of blastocysts, they
were collected on Day 7, washed in Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen, Carlsbad, CA)PVA (DPBS-PVA) and stained with 25 µg/mL of Hoechst 33342 for 10 min. After a final wash in DPBS-PVA,
blastocysts were mounted on glass slides in a drop of 100% glycerol, compressed gently with a cover slip,
and observed under a fluorescence microscope.
Immunofluorescence staining of SCNT embryos
SCNT embryos were washed three times in PBS containing 0.2% PVA, and fixed with 4%
paraformaldehyde (w/v) in PBS for 30 min. All steps were performed at room temperature unless otherwise
stated. Embryos were transferred into PBS containing 1% Triton X-100 (v/v) for 30 min. After blocking
nonspecific sites with 2% bovine serum albumin (BSA) in PBS overnight at 4 °C, embryos were then
incubated with primary antibodies (rabbit polyclonal antibody against histone H3K9 and H4K12 [Upstate
Biotechnology, Lake Placid, NY, USA], diluted 1:200) at 37°C for 3 h. Then, goat anti-rabbit fluorescein
isothiocyanate-conjugated secondary antibody (1:200; Jackson ImmunoResearch Laboratories Inc., West
Grove, PA, USA) was applied for 3 h at room temperature.
For staining of global methylation, fixed and permeabilized SCNT embryos were incubated with 50
µg/mL RNase A for 1 h at 37°C in the dark, then treated with 3 M HCl for 30 min at 37°C to denature DNA
and neutralized with 100 mM Tris for 10 min. After washing three times, samples were blocked overnight
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Post-activation treatment and embryo culture
Stock solutions of LAQ824 were dissolved in dimethyl sulfoxide (DMSO) at 20 mM and stored at -80°C.
Following electrical activation, the SCNT embryos were treated with various concentrations (0-1000 nM)
of LAQ824 for different duration (0-48 h) during IVC. After treatment, the embryos were washed three
times with fresh PZM-5 medium and then transferred into LAQ824-free medium. Cleavage and blastocyst
formation rates were evaluated on days 2 and 7, respectively, with the day of SCNT designated as day 0.
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1258
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
and incubated with primary antibodies, 5-mC mouse monoclonal antibody (mAb; Calbiochem, NA81, USA,
diluted 1:200), and secondary antibodies Alexa Fluor 488-labeled goat anti-mouse IgG (1:200). After
washing three times in PBS, DNA was counterstained with 25 µg/mL propidium iodide for 20 min.
Stained embryos were then mounted on glass slides and evaluated under an epifluorescence
microscope (TE2000-S; Nikon) with the same exposure times and adjustments. The intensities of H3K9,
H4K12 and 5-mC (green) were measured by analyzing the embryos pictures with Image J software (version
1.46r; National Institutes of Health, USA).
Analysis of gene expression by quantitative real-time PCR
Quantitative real-time PCR was performed according to a previous report [24]. Total RNAs were
extracted to analyze gene expression, using TRIzol reagent (Invitrogen), according to the manufacturer’s
protocol, and the total RNA concentration was quantified using a NanoDrop 2000 Spectrophotometer
(Thermo Fisher Scientific, Wilmington, DE, USA). Total RNA transcribed into complementary DNA (cDNA)
was produced using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). A
PCR plate (Micro-Amp Optical 96-Well Reaction Plate, Singapore) was made by adding 1 µL cDNA, 0.4 µL
(10 pmol/µL) forward primer, 0.4 µL (10 pmol/µL) reverse primer, 10 µL SYBR Premix Ex Taq (TaKaRa,
Otsu, Japan), and 8.2 µL of Nuclease-free water (NFW; Ambion, Austin, TX, USA) and then amplified on
Applied Biosystems StepOneTM Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA). The
amplification protocol included an initial denaturation step for 10 min at 95 °C followed by 40 cycles
consisting of denaturation for 15 s at 95 °C, annealing for 1 min at 60 °C, and extension for 1 min at 72
°C. All oligonucleotide primer sequences are presented in Table 3. The expression of each target gene was
quantified relative to that of the internal control gene (GAPDH) using the equation, R = 2-[△Ct sample-△Ct control]. For
ease of comparison, the average expression level of each gene from control group was set as 1.
Statistical analysis
Each experiment was repeated at least three times. The data are expressed as the mean values ±
standard error of the mean (SEM). The data were analyzed using univariate analysis of variance (ANOVA)
followed by Duncan’s multiple range test using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) statistical software.
Differences in gene expression and fluorescence intensity were compared by Student’s t-test. P < 0.05 was
considered statistically significant.
Results
LAQ824 treatment improved in vitro developmental competence of porcine SCNT embryos
We treated porcine SCNT embryos with various concentrations (0, 10, 100 and 1000 nM) of LAQ824
and statistically analyzed subsequent developmental rates at the 2-cell and blastocyst stages (Table 1).
The proportion of cleaved embryos on Day 2 was significantly lower in the group treated with 1000 nM
compared with 10 nM of LAQ824 (77.6 ± 4.2 vs. 85.0 ± 0.8, respectively, P < 0.05), but not compared with the
control and 100 nM groups. On Day 7, the 100 nM LAQ824 treated group showed a significantly increased
blastocyst formation rate compared to the control group (22.0 ± 2.8 vs. 11.9 ± 2.2, respectively, P < 0.05).
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Table 1. In vitro development of porcine SCNT embryos with different concentrations of LAQ824 for 24 h.
a,b Values with different superscript letters within a column differ significantly (P < 0.05). Experiment was
replicated at least three times
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1259
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
Table 2. In vitro development of porcine SCNT embryos with 100 nM of LAQ824 for different durations.
a,b,c Values with different superscript letters within a column differ significantly (P < 0.05). Experiment was
replicated at least three time
Table 3. Primer sequences used for real-time PCR. F, ForThen, porcine SCNT embryos were ward primer; R, Reverse primer
treated with 100 nM LAQ824 for 0, 6, 24 and
48 h (Table 2). Treatment with LAQ824 had
no effect on the subsequent embryo cleavage
rates, but treatment for 24 h significantly
improved development to the blastocyst
stage compared with the control group (29.9
± 2.9 vs. 14.2 ± 1.0, respectively, P < 0.05).
Moreover, treatment with 100 nM LAQ824
for 24 h significantly increased the total cell
number/blastocyst vs. the control group (P <
0.05).
Gene expression levels in LAQ824 treatment and control groups in SCNT-derived blastocysts
Relative expression levels of 18 genes were analyzed between the LAQ824 treatment and control
groups in SCNT-derived blastocysts using quantitative real-time PCR (Fig. 5). In histone acetylation-related
genes, LAQ824 treatment significantly increased HAT1, and decreased HDAC1, 2, 3 and 6. The levels of
mRNA expression for DNA methylation-related genes (DNMT 1, 3a and 3b) were decreased in the LAQ824
treatment group. Expression levels of development-related genes (Pou5f1, Nanog, Sox2 and GLUT1) in the
LAQ824 treatment group were significantly higher than in the control group. Transcript levels for apoptosisrelated genes (Bax, Bcl2, Caspase3 and Bak) were positively regulated in the LAQ824 treatment group.
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Detection of immunofluorescence at
various SCNT embryo development
stages
To find how LAQ824 treatment improved
the in vitro developmental competence of
porcine SCNT embryos, histone acetylation
(H3K9 and H4K12) and DNA methylation (5mC) levels of epigenetic markers were studied
at the pseudo-pronuclear, 2-cell, 4-cell and
blastocyst stages. The immunofluorescence
signals for histone acetylation (H3K9 and
H4K12) and DNA methylation (5-mC)
were positively upregulated in the LAQ824
treatment group at the pseudo-pronuclear
(Fig. 1) and 2-cell stages (Fig. 2), while the
level of H4K12 was also enhanced at the 4-cell
stage (Fig. 3). However, at the blastocyst stage
there was no significant differences between
the control and treatment groups (Fig. 4).
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1260
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
Fig. 1. Levels of H3K9,
H4K12, and 5-mC in
pseudo-pronuclear
stage embryos. (A)
Immunofluorescence
staining at the pseudo-pronuclear stage of
H3K9, H4K12, and 5-mC
(green) in the control
group and after 100 nM
LAQ824 treatment for
24 h post-activation.
Each sample was counterstained with propidium iodide (PI) to visualize DNA (red). Merged
images of H3K9, H4K12,
and 5-mC and DNA
staining are shown. The
merged images of H3K9
and DNA are yellow.
Scale bar indicates 50
μm. (B) Quantifications
of H3K9, H4K12, and
5-mC are represented
by signal intensities per
group. Fluorescence intensity was measured using Image J software 1.46r. Bars and error bars show mean values and SEM, respectively. Different asterisk above columns of the same development stage indicate statistically significant
differences (P < 0.05).
To date, somatic cells have been reprogrammed into pluripotency using various
approaches, the most common methods being induced pluripotent stem cells (iPSCs) [25-27]
or nuclear transfer into the enucleated cytoplasm of metaphase II oocytes using SCNT [2840]. Embryonic stem cells (ESCs) have greater therapeutic potential than iPSCs because they
have fewer genetic and epigenetic defects [41]. Porcine SCNT is also a promising technology,
with potential applications in regenerative medicine and xenotransplantation [2]. Therefore,
we need to address the low efficiency of porcine SCNT, which is mainly attributed to abnormal
epigenetic modifications.
HDAC inhibitors are being increasingly used in IVC treatment for improving the
efficiency of SCNT and parthenogenetic activation [42]. In this study, we sought to improve
the development of porcine SCNT embryos with LAQ824 (class I, IIa and IIb HDAC inhibitor)
treatment. We found that LAQ824 treatment (100 nM for 24 h) positively regulated epigenetic
reprogramming and gene expression in SCNT embryos. Moreover, it significantly increased
blastocyst formation rate and blastocyst quality as measured by development-related genes
and total cell numbers.
Histone acetylation, a key epigenetic factor, modifies and regulates chromatin
configuration and also plays a crucial role in nuclear reprogramming. It is well accepted that
HDACi suppress the expression of HDACs, resulting in reduced deacetylation and increased
acetylation levels. In the present study, we tested the acetylation level of SCNT embryos.
Compared to the control group, LAQ824 treatment enhanced the levels of H3K9 and H4K12
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Discussion
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1261
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
at the pseudo-pronuclear and 2-cell stages, but there was no significant difference at the
blastocyst stage. Moreover, LAQ824 treatment produced a higher level of H4K12 at the 4-cell
stage. In addition, we also detected increased gene expression related to histone acetylation
in blastocysts. The LAQ824 treatment not only upregulated gene expression of HAT1, but
downregulated gene expression of HDAC1, 2, 3 and 6. Therefore, one of the ways in which
LAQ824 treatment improved the in vitro developmental competence of porcine SCNT
embryos may be that increased histone acetylation levels, caused by promotion of HATs and
inhibition of HDAC activities, may facilitate chromatin opening. DNA methylation, as another
epigenetic marker [43], also plays an important role in the process of reprogramming
and influences in vitro developmental competence of SCNT embryos via the activity of
DNA methyltransferases (DNMTs). In this study, we revealed that LAQ824 treatment
downregulated the mRNA expression of DNMT1, 3a and 3b in blastocysts. Moreover, the
protein level of 5-mC was significantly reduced at the pseudo-pronuclear and 2-cell stages
with LAQ824 treatment, but not at the 4-cell and blastocyst stages. The EGA occurs at the
4-cell stage in porcine embryos. Accordingly, immunostaining results demonstrated that
H3K9 and 5-mC have close relationships with EGA, but H4K12 may not. In addition, these
findings illustrated that LAQ824 treatment improved epigenetic reprogramming in porcine
SCNT embryos.
To evaluate the quality of blastocysts, we measured the mRNA expression levels of
genes related to development (Pou5f1, Nanog, Sox2, CDX2, and GLUT1) and apoptosis (Bax,
Bcl2, caspase3, and Bak) in SCNT blastocysts with LAQ824 treatment.
The first distinct lineage differentiation in the mammalian embryo occurs at the
blastocyst stage; when blastomeres are segregated into inner cell mass (ICM) and
trophectoderm (TE) [44]. Pou5f1 plays an important role in determining early steps in
embryogenesis and differentiation [45]. Transcription factors, Pou5f1 and Nanog, are
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Fig. 2. Levels of H3K9,
H4K12, and 5-mC in 2-cell
stage embryos. (A) Immunofluorescence staining
at the 2-cell stage of H3K9,
H4K12, and 5-mC (green) in
the control group and after
100 nM LAQ824 treatment
for 24 h post-activation.
Each sample was counterstained with propidium iodide (PI) to visualize DNA
(red). Merged images of
H3K9, H4K12, and 5-mC and
DNA staining are shown.
The merged images of H3K9
and DNA are yellow. Scale bar indicates 50 μm. (B)
Quantifications of H3K9,
H4K12, and 5-mC are represented by signal intensities
per group. Fluorescence intensity was measured using
Image J software 1.46r. Bars
and error bars show mean
values and SEM, respectively. Different asterisk above columns of the same development stage indicate statistically significant differences (P < 0.05).
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1262
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
expressed in ICM cells and act to maintain pluripotency. However, it has been reported that
Pou5f1 is not only expressed in ICM, but also in TE of porcine blastocysts [46]. In the present
study, the expression of Pou5f1 was significantly increased in the LAQ824 treatment group.
Nanog, which is considered to be another ICM marker [47], was more highly expressed
in the LAQ824 treatment group. Sox2 in cooperation with Pou5f1 has also been shown to
maintain the pluripotent ESCs phenotype [48]; its expression level was also enhanced with
LAQ824 treatment. In contrast, CDX2 is dispensable for TE differentiation and essential for
trophectoderm stem cell self-renewal [49], however, there was no significant difference
between the control and LAQ824 treatment groups. GLUT1 is a major glucose transporter in
the ICM, which contains the ESCs; it is also expressed in TE cells [50]. The mRNA expression
of GLUT1 was enhanced by LAQ824 treatment at the blastocyst stage. In addition, there is a
relationship between blastocyst quality and apoptosis [51]. LAQ824 treatment significantly
reduced expression of pro-apoptotic genes (Bax, Caspase3 and Bak) and increased antiapoptotic gene expression (Bcl2) in blastocysts. These results demonstrated that LAQ824
treatment not only increased the expression of development-related genes, but also
improved ICM development. Therefore, LAQ824 treatment may be beneficial for supporting
ESCs. Moreover the total cell number/blastocyst and blastocyst quality were improved in the
LAQ824 treatment group by positively regulating apoptotic genes.
In conclusion, the present study indicated that 100 nM LAQ824 treatment for 24 h
post-activation, improved the in vitro developmental competence of porcine SCNT embryos
by positively regulating epigenetic reprogramming levels, and gene expression related to
histone acetylation and DNA methylation. Furthermore, LAQ824 treatment enhanced total
cell number/blastocyst and the quality of blastocysts by increasing expression of Pou5f1,
Nanog, Sox2, GLUT1 and Bcl2, and decreasing expression of Bax, Caspase3 and Bak.
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Fig. 3. Levels of H3K9,
H4K12, and 5-mC in
4-cell stage embryos.
(A) Immunofluorescence staining at the 4-cell
stage of H3K9, H4K12,
and 5-mC (green) in the
control group and after
100 nM LAQ824 treatment for 24 h post-activation. Each sample
was counterstained with
propidium iodide (PI)
to visualize DNA (red).
Merged images of H3K9,
H4K12, and 5-mC and
DNA staining are shown.
The merged images of
H3K9 and DNA are yellow. Scale bar indicates 50
μm. (B) Quantifications of
H3K9, H4K12, and 5-mC
are represented by signal
intensities per group. Fluorescence intensity was
measured using Image J
software 1.46r. Bars and
error bars show mean values and SEM, respectively. Different asterisk above columns of the same development stage indicate statistically significant differences (P < 0.05).
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1263
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
Fig. 4. Levels of H3K9, H4K12,
and 5-mC in blastocyst stage embryos. (A) Immunofluorescence staining at the
blastocyst stage of H3K9,
H4K12, and 5-mC (green) in
the control group and after
100 nM LAQ824 treatment
for 24 h post-activation. Each
sample was counterstained
with propidium iodide (PI) to
visualize DNA (red). Merged
images of H3K9, H4K12, and
5-mC and DNA staining are
shown. The merged images
of H3K9 and DNA are yellow.
Scale bar indicates 50 μm.
(B) Quantifications of H3K9,
H4K12, and 5-mC are represented by signal intensities
per group. Fluorescence intensity was measured using
Image J software 1.46r. Bars
and error bars show mean
values and SEM, respectively.
Fig. 5. Messenger
RNA (mRNA) expression levels (mean ±
SEM) of (A) histone
acetylation, (B) DNA
methylation, (C) development and (D)
apoptosis-related
genes in blastocysts
derived from fusion couplets treated
with 100 nM LAQ824
for 24 h. Within the
same mRNA group,
bars with different
superscript letters
are significantly different (P < 0.05).
Cas3, Caspase3.
SCNT (somatic cell nuclear transfer); HAT (histone acetyltransferases); HDAC (histone
deacetylase); EGA (embryonic genome activation); COCs (cumulus-oocyte complexes);
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Abbreviations
Physiol Biochem 2017;41:1255-1266
Cellular Physiology Cell
© 2017 The Author(s). Published by S. Karger AG, Basel
DOI: 10.1159/000464389
and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
1264
Jin et al.: Effects of LAQ824 on Porcine Cloned Embryos
IVM (in vitro maturation); IVC (in vitro culture); ITS (insulin transferrin selenium); EGF
(epidermal growth factor); hCG (human chorionic gonadotropin); eCG (equine chorionic
gonadotropin); DMEM (Dulbecco’s modified Eagle's medium); FBS (fetal bovine serum);
PBS (phosphate-buffered saline); TALP (Tyrode's albumin lactate pyruvate); PZM-5 (porcine
zygote medium-5); DMSO (dimethyl sulfoxide); cDNA (Complementary DNA).
Acknowledgements
This study was supported by the Korea Institute of Planning and Evaluation for
Technology in food, agriculture, forestry and fisheries (#311011-05-4-SB010, #114059-032-SB010), Ministry of Trade, Industry & Energy (#10048948), National Research Foundation
(2016M3A9B6903410), China Scholarship Council (CSC, No. 2015-3022), Research Institute
for Veterinary Science, TS Corporation and the BK21 plus program.
Disclosure Statement
The authors have no conflict of interest to disclose.
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and Biochemistry Published online: March 07, 2017 www.karger.com/cpb
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